Maternal Neutralizing Antibody and Transmission of Hepatitis C Virus to Infants

Hepatitis C virus (HCV) is transmitted to a minority of children born to HCV-infected mothers [1–3]. The risk of mother-to-child transmission (MTCT) of HCV is increased if the woman is also infected with HIV-1 [3]. HIV increases the abundance of HCV RNA detected in plasma (HCV load), and a higher maternal HCV load has been associated with increased MTCT of HCV [2]. However, how HIV infection enhances HCV transmission, whether or when HCV is transmitted across the intact placenta, and why the majority of children born to HCV-infected women remain uninfected are unknown.

In the months after acute HCV infection, adaptive immune responses are detectable that are sufficient to clear specific viral variants from blood (and drive evolution) but that are inadequate to eradicate infection [4, 5]. The recent development of HCV pseudoparticles (HCVpps) has allowed characterization of neutralizing antibodies (nAbs) specific for HCV envelope glycoproteins [6, 7]. We and others have found marked interpersonal differences in the titers and kinetics of HCV nAbs [4, 6, 8–11]. We have also found that HIV infection reduces HCV-specific antibody titers [12]. In the present investigation, we tested the hypothesis that low maternal HCV nAb titers among HCV/HIV-coinfected mothers is associated with MTCT of HCV.

Methods. Study subjects were participants in the Women and Infants Transmission Study (WITS) [2]. From 1989 to 2005, HIV-infected women were enrolled at institutions in New York City, Chicago, Boston, Houston, and San Juan, Puerto Rico. Women could enroll at any time during pregnancy and were examined at entry and at structured time points during pregnancy, at delivery, and thereafter. At each visit, a physical examination was performed and a medical history obtained; venous blood was collected, and components were stored at −70°C. At delivery or shortly thereafter and at 1, 2, 4, 6, 9, 12, 15, 18, 24, 30, and 36 months of age, infants were examined and venous blood was collected. According to protocol, children were not breast-fed. The study was approved by the institutional review board at each participating center. Written informed consent was obtained from each participating woman.

MTCT of HCV was assessed in HCV antibody-positive mother-infant pairs who had at least 6 months of infant follow-up. Previously, HCV infection was documented in 12 infants with follow-up until September 1994 [13]. Testing of additional mothers and infants enrolled through 2005 revealed another 5 HCV-infected infants.

To test the hypothesis that lower titers of HCV nAbs increase the risk of infant HCV infection, a nested case-control study was conducted among mothers of HCV-infected and HCV-uninfected infants. Each HCV case patient was matched with up to 3 mothers of HCV-uninfected infants by age (±5 years), CD4⁺ lymphocyte count at delivery (>500, 351–500, 200–350, and <200 cells/mm³), transmission risk (assessed on the basis of mode of delivery [scheduled cesarean section, unscheduled cesarean section, or vaginal birth] and duration of time from rupture of membranes to delivery [>4 h or ≤ 4 h]), and time of cohort enrollment (before 1 March 1994, on or between 1 March 1994 and 31 July 1996, and after 31 July 1996). If available, plasma from 2 time points was tested, one sample from the time of delivery and another from either 2 or 12 months postpartum.

Antibodies to HCV were detected using an commercially available ELISA (HCV 2.0 or greater; Ortho Diagnostic Systems) in heparinized plasma. The concentration of HCV RNA in plasma was ascertained using a commercially available assay (Amplicor Monitor HCV Assay; Roche Diagnostic Systems).

HCV nAb titers were assessed in maternal plasma at the Viral Hepatitis Laboratory at Johns Hopkins. The testing laboratory was blinded to the transmission status of the subjects. HCVpps were generated by cotransfection of 293T cells (ATCC) with pNL4-3.Luc.R⁻E⁻ plasmid containing the env-defective HIV proviral genome and an expression plasmid containing HCV glycoproteins (strain H77), as described elsewhere [4, 7]. Virus-containing medium was collected at 48 and 72 h, pooled, and stored in aliquots at -80°C. Experiments were done with equivalent amounts of HCVpps, as assessed by HIV p24 antigen ELISA (Aalto Bio Reagents). For neutralization experiments, 2-fold dilutions of heat-inactivated plasma or serum, starting at 1:50, were incubated with HCVpps for 1 h at 37°C and added to Hep3B hepatoma cells (ATCC) for 5 h, after which the virus-containing medium was removed. After 72 h, cells were lysed with 50 µL of Cell Culture Lysis Reagent (Promega), and luciferase activity was detected by the addition of 50 µL of Luciferase Assay Reagent (Promega), measured for 5 s in a luminometer (Berthold Technologies). Pseudotype infection resulting in luciferase activity was measured in terms of relative light units (RLUs) in the presence of test plasma (RLU_test) versus that in the presence of an HCV-negative normal human serum specimen (RLU_control) at the same dilution. Percent neutralization was calculated as 100 × [1− (RLU_test/RLU_control)], and end-point neutralization titers were reported as the dilution of plasma that resulted in ≥50% inhibition of HCVpp infectivity (ID₅₀). If the ID₅₀ values for paired visits from the same mother were within 1 dilution, neither sample was retested. Discrepant samples were retested to confirm the ID₅₀ at each visit or to clarify unclear results. Assays were repeated to confirm results for those specimens derived from only a single visit.

Negative control pseudoparticles expressing no envelope protein produced RLU values 5 orders of magnitude lower than those for HCVpps. Positive control pseudoparticles expressing the murine leukemia virus glycoprotein were insignificantly neutralized (consistently <35%) by maternal plasma samples at a 1:50 dilution, demonstrating that any antiretroviral drugs present in maternal samples did not inhibit pseudoparticle infectivity.

For statistical analysis, case and control groups were compared according to matching criteria and other factors. The distribution of maternal HCV nAb titers was assessed, and reciprocal values were log₁₀ transformed to approximate a normal distribution. Titers that were below the level of detection were assigned an ID₅₀ value of 1:25 for analysis. The rank order of titers was compared in mothers according to infant HCV status to evaluate the statistical significance of differences in distributions. Then, a conditional logistic regression model of HCV MTCT was constructed to assess the contribution of HCV nAb titer along with other factors, such as maternal plasma HCV RNA concentrations. Analyses were performed using the PROC PHREG procedure in SAS software (SAS Institute). P < .05 was considered to indicate statistical significance.

Results. We studied plasma from 16 of 17 mothers of HCV-infected infants (one had insufficient sample). In addition, we evaluated plasma from 47 mothers of HCV-uninfected infants. HCV-transmitting mothers were similar to nontransmitting mothers with respect to age, race, use of illicit drugs during pregnancy, CD4⁺ lymphocyte count, mode of delivery, and gestational age at delivery (table 1). A greater proportion of HCV-infected infants than HCV-uninfected infants were also infected with HIV, but this difference did not reach statistical significance.

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Figure 1

Distribution of hepatitis C virus (HCV) neutralizing antibody titers, by HCV transmission status. Maternal plasma from 2 visits (one sample from the time of delivery and another from either 2 or 12 months postpartum; note that, because of sample availability, 4 mothers had serum from only a single visit tested) was assessed for its ability to neutralize heterologous HCV pseudoparticles expressing the envelope glycoproteins from strain H77. Points represent log₁₀-transformed reciprocal ID₅₀ titers for each mother, generated by combining assay results from both visits and taking the median value. Values are plotted on a log scale, with the nominal values shown on the Y-axis; horizontal lines represent the median value for each group. ID₅₀ titers for mothers who transmitted HCV to their infant (n = 16) did not significantly differ from those for nontransmitting mothers (n = 47) (P = .23).

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Table 1

Characteristics of hepatitis C virus (HCV)/HIV-coinfected mothers, according to whether HCV was transmitted to the infant.

Titers of nAbs were reproducible in paired visits from maternal samples. Of the 63 case and control patients, 59 mothers had paired visits (for 4 subjects, plasma was available only from a single visit). Of these 59 subjects, 51 (86.44%) had ID₅₀ values that were identical (35 subjects [59.32%]) or that differed by 1 dilution (16 subjects [27.12%]). The ID₅₀ values of the remaining 8 subjects (13.56%) differed by ≥2 dilutions. Because the paired visits showed general agreement, we used the median ID₅₀ value of all neutralization experiments from each mother as a single representative neutralization titer. Including all mothers, ID₅₀ titers ranged from undetectable to 1:12,800, with a median of 1:100. A total of 13 mothers (20.63%) had undetectable neutralization titers at a 1:50 dilution of plasma.

An HCV MTCT model was constructed to examine the role played by HCV nAbs. As previously published, the odds of HCV transmission were increased 1.05-fold for each 1 × 10⁵ IU/mL increase in HCV load (P = .03) (table 2) [2]. HCV nAbs were detected in 15 transmitting mothers (93.75%) and in 35 nontransmitting mothers (74.47%). The median (range) of nAb titers in women who transmitted HCV, 1:125 (undetectable to 1:12,800), was not significantly different from that in women who did not transmit HCV, 1:100 (undetectable to 1:3200), (P > .05) (table 2 and figure 1). Multivariate analysis detected no interaction or effect modification in the associations between transmission status, maternal HCV nAb titer, and HCV load.

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Table 2

Association of maternal and infant factors with mother-to-infant transmission of hepatitis C virus (HCV).

Discussion. The results of this investigation do not confirm our hypothesis that HCV nAb titers are associated with MTCT of HCV. Because this is the first study that we are aware of to investigate the role played by nAbs in the transmission of HCV infection, there is a limited degree to which we can discuss how our data compare with others. More work is clearly needed to understand why HCV-infected mothers transmit infection to some, but not most, infants.

There are several reasons why HCV nAb titers might not alter the frequency of MTCT of HCV. Broadly reactive nAbs are readily detected in chronically infected persons, yet the virus persists [8, 11, 14]. Autologous neutralization studies using HCVpps and serum from prototype H77 infection showed ongoing viral escape from the nAb response during chronic infection [15]. We have recently corroborated these findings in multiple acutely infected subjects, suggesting that serum nAbs recognize previous, but not concurrent, viral sequences because of continuing evolution of viral envelope sequences (unpublished data). In line with these results, it follows that nAbs that are ineffective at eliminating circulating virus in the mothers may be equally ineffective at preventing infection of the infant. Likewise, nAbs may efficiently block transmission of some, but not all, variants, allowing infection.

The high variability of HCV sequences presented a technical limitation to our study. The assay measures the extent to which the mother's plasma neutralizes infection of cells by HCVpps expressing a prototype genotype 1a HCV envelope sequence (H77). Divergence between the H77 sequence and those of the mother's previous and current circulating viruses—particularly differences in genotype or subtype—may have resulted in an underestimation of nAb titers. In the setting of chronic HCV monoinfection, during which broadly reactive, high-titer nAbs are readily detectable, the use of heterologous HCVpps should provide an adequate representation of nAb levels. However, we have shown that coinfection with HIV lowers HCV antibody titers [12], potentially limiting our detection of nAb in this study. Of note, the median titer for all mothers was 1:100, with 20.63% showing undetectable nAbs. These are much lower than titers we and others have obtained from subjects with chronic HCV monoinfection. Therefore, it is possible that the effect of HIV infection on antibody production diminished our ability to test the hypothesis in this context.

We and others have found that the risk of MTCT of HCV is increased in mothers with a higher HCV load and in those with concomitant HIV infection. In some, but not most, studies, the mode of transmission also appears to be a factor. A strength of the present investigation of nAbs was our ability to control for or examine potential confounding factors. Because HIV infection and age may affect the risk of HCV transmission and nAb titer, it is important that we adjusted for these factors by matching [2, 12]. We did not match on the basis of HCV load in order to investigate whether nAb titer contributed to viral load and its established relationship with transmission risk. A significant correlation was detected between increased nAb titers and increased HCV RNA levels (Spearman's rank correlation coefficient, 0.46; P < .001). Because higher HCV loads are associated with increased HCV transmission risk, this finding provides a possible explanation for higher nAb titers in HCV-transmitting mothers than in HCV-nontransmitting mothers (ID₅₀, 1:125 vs. 1:100).

This investigation was restricted to HIV-infected women, who were found to have remarkably low titers of nAb overall. Transmission of HCV from HIV-uninfected women is rare, limiting opportunities to investigate the hypothesis in that setting. Thus, we cannot exclude the possibility that HCV nAbs are sufficient, but not necessary, to prevent MTCT of HCV, and it remains possible that the low-titer nAbs we observed have a small effect on HCV transmission that would have been detected in a larger study. Nonetheless, these data clearly indicate that maternal nAb titers measured using a heterologous strain do not substantially contribute to the prevention of MTCT of HCV among HCV/HIV-coinfected mothers. Additional work is needed to understand the biological basis of HCV transmission.

WITS. Principal investigators, study coordinators, program officers, and funding for WITS include the following: Clemente Diaz and Edna Pacheco-Acosta (University of Puerto Rico, San Juan; grant U01 AI 34858); Ruth Tuomala, Ellen Cooper, and Donna Mesthene (Boston/Worcester Site, Boston, Massachusetts; grant 9U01 DA 15054); Phil LaRussa and Alice Higgins (Columbia Presbyterian Hospital, New York, New York; grant U01 DA 15053); Sheldon Landesman, Hermann Mendez, Edward Handelsman, and Ava Dennie (State University of New York, Brooklyn; grant HD-3–6117); Kenneth Rich and Delmyra Turpin (University of Illinois at Chicago, Chicago; grant U01 AI 34841); William Shearer and Norma Cooper (Baylor College of Medicine, Houston, Texas; grant U01 HD 41983); Joana Rosario (National Institute of Allergy and Infectious Diseases, Bethesda, Maryland); Kevin Ryan (Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland); Vincent Smeriglio and Katherine Davenny (National Institute on Drug Abuse, Bethesda, Maryland); and Bruce Thompson (Clinical Trials and Surveys, Baltimore, Maryland; grant N01 AI 085339). The Scientific Leadership Core included Kenneth Rich (principal investigator) and Delmyra Turpin (study coordinator) (grant U01 AI 50274-01). Additional support was provided by local clinical research centers, as follows: Children's Hospital Boston, Boston, Massachusetts (NIH grant GCRC RR02174); Baylor College of Medicine, Houston, Texas (NIH grant GCRC RR00188); and Columbia University, New York, New York (NIH grant GCRC RR00645).

• Received April 15, 2008.
• Accepted June 27, 2008.

• © 2008 by the Infectious Diseases Society of America